The present invention relates to DNA encoding levodione reductase, an expression vector containing the DNA, a recombinant vector containing the DNA, a microorganism into which the DNA has been introduced, and a method for producing (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (hereinafter referred to as actinol) from (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (hereinafter referred to as levodione) using the microorganism. Actinol is a useful chiral building block of naturally occurring optically active compounds such as zeaxanthin.
European Patent Application No. 98115564.1, filed on Aug. 19, 1998, discloses a process for the manufacture of actinol, which involves contacting levodione with a microorganism selected from the group consisting of microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter, which is capable of selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture. In this process, one of the most effective strains was Corynebacterium aquaticum AKU611 (FERM BP-6448), which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, in the name of F. Hoffmann-La Roche A G of Grenzacherstrasse 124, CH-4070 Basel, Switzerland on Aug. 4, 1998, under the Budapest Treaty.
European Patent Application No. 99102037.1, filed on Feb. 1, 1999, discloses an enzyme, levodione reductase, that acts on levodione to produce actinol, which was isolated from Corynebacterium aquaticum AKU611 (FERM BP-6448). This enzyme is characterized by the following physico-chemical properties: 1) The levodione reductase catalyzes the regio- and stereoselective reduction of levodione to actinol. 2) The relative molecular mass of the enzyme is estimated to be 142,000-155,000xc2x110,000 Da, consisting of four homologous subunits having a molecular mass of 36,000xc2x15,000 Da. 3) The optimum temperature is 15-20xc2x0 C. at pH 7.0 and the optimum pH is 7.5. 4) The enzyme requires NAD+ or NADH as a cofactor and is highly activated by monovalent cations, such as K+, Na+, Cs+, Rb+, and NH4+. 
An object of the present invention is a DNA sequence encoding for an enzyme, levodione reductase, which is useful for the preparation of actinol, an important intermediate in the production of zeaxanthin.
The isolated DNA sequence may be more specifically characterized by the following:
(a) the nucleotide sequence codes for the enzyme having the amino acid sequence shown in SEQ ID No.: 1, or
(b) the nucleotide sequence codes for a variant of the enzyme selected from (i) an allelic variant, or (ii) an enzyme having one or more amino acid additions, insertions, deletions and/or substitutions, but still having the same type of enzymatic activity.
The isolated DNA sequence mentioned above may be derived from a gene of Corynebacterium aquaticum and selected from
(i) the nucleotide sequence shown in SEQ ID No.: 2;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes a levodione reductase having the same amino acid sequence as that encoded by SEQ ID No: 2, or
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from (i) or (ii) under standard hybridizing conditions.
Another object of the present invention is a recombinant DNA which codes for levodione reductase and which can be obtained by genetic recombination of the isolated DNA described above. Such a recombinant DNA may preferably be in the form of a vector. The recombinant DNA may contain regulatory regions, such as promoters and terminators, as well as an open reading frame of the gene described above.
A further object of the invention is a recombinant organism consisting of a host organism transformed with the recombinant DNA. A preferred form of the recombinant DNA is a vector. The host organism transformed with the recombinant DNA may be useful in improving the process of actinol production.
Another object of the present invention is a method for the biological production of actinol that includes introducing a recombinant DNA, as described above, into an appropriate host organism, and cultivating the resulting recombinant organism in the presence of levodione as a substrate.
Accordingly, the invention provides an isolated polynucleotide encoding a polypeptide having levodione reductase activity.
Another embodiment of the invention is a vector or a plasmid containing a polynucleotide sequence encoding a polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
A further embodiment of the invention is a microorganism transformed or transfected with a polynucleotide sequence which encodes a polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
Another embodiment of the invention is an isolated polypeptide having levodione reductase activity wherein the polypeptide has the properties as set forth above.
A further embodiment of the invention is a process for producing a polypeptide having levodione reductase activity. This process includes culturing a microorganism transformed or transfected with a polynucleotide encoding a polypeptide having levodione reductase activity in nutrient media and isolating the polypeptide produced by the microorganism.
The present invention also provides a process for producing actinol. This process includes contacting levodione with a polypeptide having levodione reductase activity.
As used herein, xe2x80x9clevodione reductasexe2x80x9d is a polypeptide that catalyzes, regio- and stereoselectively, the conversion of levodione to actinol in the presence of NADH. European Patent Application No. 99102037.1, filed on Feb. 1, 1999, discloses the physico-chemical properties of the levodione reductase. The levodione reductase of the invention can be prepared by cultivating an appropriate microorganism in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the levodione reductase from the cell-free extract. Many species have been found to catalyze this conversion, including the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter. A preferred strain for the enzyme is Corynebacterium aquaticum AKU611 (FERM BP-6448).
As used herein, an xe2x80x9callelexe2x80x9d or xe2x80x9callelic variantxe2x80x9d is an alternative form of a gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times, in a given sequence.
A xe2x80x9cvariantxe2x80x9d of levodione reductase, as used herein, is an amino acid sequence that is altered by one or more amino acids. The variant may have xe2x80x9cconservativexe2x80x9d changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have xe2x80x9cnon-conservativexe2x80x9d changes, e.g., replacement of glycine with tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. A xe2x80x9cdeletionxe2x80x9d, as used herein, refers to a change in either the amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent. An xe2x80x9cinsertionxe2x80x9d or xe2x80x9cadditionxe2x80x9d, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule. A xe2x80x9csubstitutionxe2x80x9d, as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
As used herein, xe2x80x9cexpression vectorxe2x80x9d includes vectors that are capable of expressing DNA sequences contained therein, where such sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not explicitly stated, that expression vectors must be replicable in the host organisms either as episomes or as an integral part of chromosomal DNA. Clearly, a lack of replication would render them effectively inoperable. Thus, xe2x80x9cexpression vectorxe2x80x9d is also given a functional definition. Generally, expression vectors of utility in DNA recombinant techniques are in the form of xe2x80x9cplasmidsxe2x80x9d. xe2x80x9cPlasmidsxe2x80x9d refer to either circular double stranded DNA molecules or circular single stranded DNA molecules, containing an origin of replication derived from a filamentous bacteriophage. These DNA molecules, in their vector form, are not linked to the chromosomes. Other effective vectors commonly used are phage and non-circular DNA. In the present specification, xe2x80x9cplasmidxe2x80x9d and xe2x80x9cvectorxe2x80x9d are often used interchangeably. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or subsequently become, known.
xe2x80x9cRecombinant host cellsxe2x80x9d, xe2x80x9chost cellxe2x80x9d, xe2x80x9ccellsxe2x80x9d, xe2x80x9ccell culturesxe2x80x9d and so forth, are used interchangeably herein to designate individual cells, cell lines, cell cultures, and harvested cells, which have been or are intended to be transformed with the recombinant vectors of the invention. The terms also include the progeny of the cells originally receiving the vector.
The terms xe2x80x9cDNA,xe2x80x9d xe2x80x9cnucleotide sequencexe2x80x9d and xe2x80x9cpolynucleotidexe2x80x9d or xe2x80x9cpolynucleotide sequencexe2x80x9d are used interchangeably throughout and are intended to have the same meaning unless otherwise indicated. Likewise, the terms xe2x80x9camino acid sequencexe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used interchangeably throughout and are intended to have the same meaning unless otherwise indicated.
xe2x80x9cTransformedxe2x80x9d or xe2x80x9ctransformationxe2x80x9d refers to any process for altering the DNA content of the host.
As used herein, the phrase xe2x80x9cstandard hybridizing conditionsxe2x80x9d refers to conditions under which the person skilled in the art obtains a specific signal. The conditions may range from low stringency conditions (6xc3x97SSC, 50xc2x0 C.; overnight; washing in 6xc3x97SSC at room temperature) to preferred medium stringency conditions (6xc3x97SSC; 65xc2x0 C.; overnight; washing in 6xc3x97SSC at 30xc2x0 C.) to the most preferred high stringency conditions (6xc3x97SSC; 75xc2x0 C.; overnight; washing two times in 6xc3x97SSC at 37xc2x0 C.). Alternatively, the degree of similarity can be determined by the percentage of homology. For the determination of homology the two sequences to be compared are aligned to each other by a suitable computer program. Using the standard conditions of the program, the claimed sequences have a percentage of homology of at least 40%, preferably, at least 60%, and more preferable at least 80%, with the sequences disclosed in the present application.
Amino acids are identified by either their single-letter or three-letter designations:
The following is a list of commercial suppliers for materials used in this invention:
a. Invitrogen: 1600 Faraday Avenue, Carlsbad, Calif. 92008, USA
b. Amersham Pharmacia Biotech: SE-751 84 Uppsala, Sweden
c. Toyobo: 2-2-8 Dojimahama, Kita-ku, Osaka, Japan
d. Takara Shuzo: 2-15-10 Nihonbashi, Chuo-ku, Tokyo, Japan
e. Promega: 2800 Woods Hollow Road, Madison, Wis., USA
f. BIO101: 2251 Rutherford Rd., Carlsbad, Calif. 92008, USA
g. PE Biosystems: 850 Lincoln Center Drive, Foster City, Calif. 94404, USA
h. Shimadzu: 1 Nishinokyo, Kuwabaracho, Nakagyo-ku, Kyoto, Japan
i. Shinwa Chemical Industries: 50 Keishocho, Fushimi-ku, Kyoto, Japan
j. Wako Pure Chemicals: 3-1-2 Doshoumachi, Chuo-ku, Osaka, Japan
k. Oriental Yeast: 3-6-10 Shodosawa, Itabashi-ku, Tokyo, Japan
l. Amano Pharmaceuticals: 1-2-7 Nishiki, Naka-ku, Nagoya, Japan
The gene encoding levodione reductase is a DNA encoding a polypeptide having the enzyme activity of converting levodione to actinol. A typical example of this gene is a levodione reductase gene which can be cloned from Corynebacterium aquaticum AKU611 (FERM BP-6448). This DNA contains a nucleotide sequence coding for a polypeptide containing the amino acid sequence as shown in SEQ ID No.: 1.
The DNA sequence may be cloned from a strain of Corynebacterium aquaticum AKU611 (FERM BP-6448), or another related organism and thus, may be an allelic or species variant of the levodione reductase encoding region of the DNA sequence. Also included within the scope of the present invention is a derivative of the DNA sequence with additions, insertions, deletions and/or substitutions of different nucleotides resulting in a polypeptide that encodes the same or a functionally equivalent levodione reductase. For example, the derivative may contain from 1-100, preferably 1-50, more preferably 1-10 to 1-5 additions, insertions, deletions and/or substitution of nucleotides, so long as the derivative encodes a polypeptide have levodione reductase activity. The encoded protein may also contain addition, deletions, insertions and/or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent levodione reductase. For example, the derivative protein may contain from 1-100, preferably 1-50, more preferably 1-10 to 1-5 additions, insertions, deletions and/or substitution of amino acids so long as the derivative still retains its function (i.e., levodione reductase activity).
The levodione reductase gene product, i.e. the levodione reductase of the present invention, has, as described above, an enzyme activity to convert levodione to actinol. The gene for such an enzyme activity has not been previously described. However, by using the levodione reductase gene, it is possible to confer on a microorganism, such as E. coli, the ability to convert levodione to actinol.
The present invention provides an isolated DNA sequence that codes for an enzyme, levodione reductase, which is involved in the conversion of levodione to actinol. The DNA can include genomic DNA which contains regulatory sequences such as promoters and terminators that are involved in the expression of the gene of interest, and a cDNA which contains an open reading frame flanked between short fragments in its 5xe2x80x2- and 3xe2x80x2-untranslated region.
The levodione reductase gene, the recombinant expression vector, and the recombinant organisms utilized in the present invention may be obtained by the following; steps:
(1) Isolating chromosomal DNA from a microorganism that can provide the levodione reductase of the present invention and constructing a gene library with the chromosomal DNA.
(2) Cloning the levodione reductase gene from chromosomal DNA by colony- or plaque-hybridization, PCR cloning, Southern-blot hybridization, and the like.
(3) Determining the nucleotide sequence of the levodione reductase gene by conventional methods, and constructing recombinant expression vectors that contain and efficiently express the levodione reductase gene.
(4) Constructing recombinant organisms carrying the levodione reductase gene on recombinant expression vectors or in chromosomes by transformation, transduction, transconjugation and electroporation.
The techniques used to isolate or clone DNA encoding the levodione reductase of the present invention are known in the art and include the isolation from genomic DNA. The cloning of the DNA sequence of the present invention from genomic DNA can be effected, for example, using degenerate polymerase chain reaction (PCR).
To clone the levodione reductase gene, knowledge of the amino acid sequence of levodione reductase may be necessary. Levodione reductase protein may be purified and a partial amino acid sequence may be determined by conventional methods (Biosci. Biotechnol. Biochem. 62, 280-285, (1998)). Determination of the complete amino acid sequence is not necessary. Once suitable amino acid sequences have been identified, oligonucleotides for use as PCR primers are synthesized on the basis of information on the partial amino acid sequences. The primers used for cloning the levodione reductase gene by PCR are based on the amino acid sequence of the internal peptide fragments of the purified levodione reductase from the genera including, for example, Corynebacterium, Cellulomonas, Planococcus and Arthrobacter, and in the most preferred embodiment, from Corynebacterium aquaticum AKU611 (FERM BP-6448). A DNA fragment (i.e., a partial DNA sequence) for levodione reductase is generated by PCR amplification using the primers and a template of Corynebacterium aquaticum chromosomal DNA. The amplified DNA fragment can then be used as a probe to clone a genomic fragment coding for the whole levodione reductase of Corynebacterium aquaticum AKU611 (FERM BP-6448).
An entire gene containing its coding region as well as its regulation region, such as a promoter or terminator, can be cloned from a chromosome by screening a genomic library with a labeled probe. The genomic library should be constructed in phage or plasmid vectors in an appropriate host. The probe used to screen the library should be a partial DNA fragment obtained by PCR, as described above, that has been labeled. Generally, an E. coli vector, a phage vector (e.g., xcex phage vector), a plasmid vector, or a yeast vector, and E. coli as a host strain, are used in the construction of a library and for subsequent genetic manipulations, such as a sequencing, restriction digestion, ligation, and the like. Identification of desired clones from the plasmid or phage library is best effected by selecting a probe, such that the desired gene will hybridize to the probe under high stringency conditions.
A genomic library of Corynebacterium aquaticum AKU611 (FERM BP-6448) was constructed in pYES2. The PCR-amplified fragment used as a probe was labeled with horseradish peroxidase (xe2x80x9cHRPxe2x80x9d), according to the supplier""s protocol (Amersham Pharmacia Biotech), instead of a conventional 32P labeling method. A genomic library constructed from the chromosome of Corynebacterium aquaticum AKU611 (FERM BP-6448) was screened with an HRP-labeled DNA fragment, which had a portion of the gene of interest, as a probe. Hybridized colonies were picked up and used for further study. After the isolation of positive colonies, the insert fragments were subcloned into an appropriate sequencing vector. The insert fragments were then subcloned into a pUC18 vector.
The nucleotide sequence of the target gene can be determined by a well-known sequencing method such as the dideoxy chain-termination method (Proc. Natl. Acad. Sci. USA, 74, 5463-5467, (1977)).
The isolated DNA sequence of the present invention may also be used to identify and clone DNA encoding a polypeptide having levodione reductase activity from other strains of different genera or species according to methods well known in the art.
The present invention also includes a recombinant DNA, preferably a vector and/or plasmid, containing a sequence coding for levodione reductase. The recombinant DNA vector and/or plasmid may contain the regulatory regions, such as promoters and terminators, as well as open reading frames of the above mentioned DNA.
Methods which are well known to those skilled in the art may be used to construct expression vectors containing a sequence encoding levodione reductase and appropriate transcriptional and translational regulatory elements, including all components which are necessary or advantageous for expression of the coding sequence as described in Ausubel F. M. et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y. (1989). Specific initiation and termination signals, may also be used to achieve more efficient translation of sequences encoding levodione reductase.
An isolated DNA sequence encoding levodione reductase may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleotide sequence encoding the levodione reductase prior to its insertion into a vector may be desirable, or necessary, depending on the expression vector. The techniques for modifying nucleotide sequences utilizing cloning methods are well known in the art.
A variety of expression vector/host systems may be utilized to contain and express sequences encoding levodione reductase. These systems include, for example, microorganisms, such as bacteria, transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; plant cell systems transformed with viral expression vectors or with bacterial expression vectors; or animal cells. An expression vector is selected according to the use intended for the levodione reductase. For example, when large quantities of levodione reductase are needed, vectors that direct high level expression of the introduced DNA sequence may be used. Such vectors include, for example, the E. coli cloning and expression vectors, such as pBluescript II and pUC18.
The host cell, which is transformed with the DNA sequence coding for levodione reductase, may be either eukaryotic or prokaryotic. The choice of a host cell may, to a large extent, depend on the gene encoding the polypeptide and that gene""s source. Suitable prokaryotic host cells include bacterial cells, such as E. coli, which are used to provide for the high level expression of protein. In order to overexpress an enzyme of interest, promoter systems suitable for high level expression may be used. Such promoters include, for example, the lac or T7 expression systems.
The present recombinant DNAs, vectors, or plasmids may be used to transform a host organism. The recombinant organism obtained is capable of overexpressing the DNA sequence encoding levodione reductase. Thus, the host organism transformed with the recombinant DNA is useful in the production process of actinol. Accordingly, the present invention also includes recombinant organisms and transformed host cells.
A method for producing levodione reductase is also provided. This method includes culturing the recombinant organism of the present invention under conditions conducive to the production of the enzyme. Host cells transformed with nucleotide sequences encoding levodione reductase may be cultured under conditions suitable for the expression and recovery of the protein from a cell culture.
The recombinant organism of the present invention may be cultured in nutrient medium containing saccharides, such as glucose and sucrose, alcohols, such as ethanol and glycerol, fatty acids, such as oleic acid and stearic acid or esters thereof, or oils, such as rapeseed oil and soybean oil, as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran, yeast extract, and the like, as nitrogen sources; magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, and the like, as inorganic salt sources; and malt extract, meat extract, and the like, as other nutrient sources. Cultivation of the recombinant organism can be carried out aerobically or anaerobically, preferably for a period of 1 to 7 days at a medium pH of 3 to 9 and a cultivation temperature of 10 to 40xc2x0 C.
The levodione reductase produced by a recombinant cell of the present invention may be secreted or contained intracellularly depending on the sequence and/or the vector used. The levodione reductase may then be isolated from the culture medium, or the host cell, by conventional procedures.
The present invention also provides a process for the isolation and purification of levodione reductase from the recombinant cells after cultivation as follows:
(1) Cells are harvested from the liquid culture broth by centrifugation or filtration.
(2) The harvested cells are washed with water, physiological saline, or a buffer having an appropriate pH.
(3) The washed cells are suspended in the buffer solution and disrupted by means of a homogenizer, sonicator, French press, or treatment with lysozyme, and the like, to give a solution of disrupted cells.
(4) The levodione reductase is isolated and purified from the cell-free extract of disrupted cells.
Following confirmation of enzyme activity, the expressed levodione reductase protein may be used for raising antibodies to the purified enzyme. The antibody may then be used for characterizing the expression of the corresponding enzyme in a strain improvement study, an optimization study of culture conditions, and the like.
The following examples are provided to further illustrate methods of preparation of the compositions of the present invention, as well as certain physical properties and uses thereof. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
The following materials and methods were employed in the Examples described below. All % are % (wt) unless otherwise noted.
Strains
Corynebacterium aquaticum AKU611 (FERM BP-6448)
E. coli DH5xcex1: [xcfx8680xcex4lacZxcex94M15, Fxe2x88x92, xcexxe2x88x92, xcex94(lacZYA-argFV169), hsd R17(rKxe2x88x92, mK+), recA1, endA1, supE44, deoR, thi-1, gyrA96, relA1] (Toyobo, Osaka, Japan).
E. coli JM109: [recA1, endA1, gyrA96, thi, hsdR17(rKxe2x88x92, mK+), mcrB+, supE44, relA1, xcex94(lac-proAB), Fxe2x80x2(traD36, proAB, laclq, lacZxcex94M15), xcexxe2x88x92] (Toyobo, Osaka, Japan).
Vectors
pYES2 (Invitrogen)
pGEM-T (Promega)
pUC18 (Toyobo)
Media
Corynebacterium aquaticum AKU611 (FERM BP-6448) was cultured aerobically at 30xc2x0 C. for 20 hours in a medium (pH 7.0) containing 1% glucose, 1.5% peptone, 0.3% K2HPO4, 0.1% yeast extract, 0.2% NaCl, and 0.02% MgSO4 7H2O. E. coli transformants containing the Corynebacterium aquaticum AKU611 (FERM BP-6448) levodione reductase gene were grown in Luria-Bertani medium (LB medium) consisting of 10 g of tryptone, 10 g of sodium chloride, and 5 g of yeast extract (pH 7.2)/liter, or in M9 medium (page A-3, Molecular Cloning, 1989, Cold Spring Harbor Laboratory Press) supplemented with casamino acids.
Enzymes and Chemicals
Restriction endonucleases and other DNA-modifying enzymes were obtained from Takara Shuzo and Toyobo. Ex taq, a Taq DNA polymerase, and Ex taq buffer were purchased from Takara Shuzo. ECL direct nucleic acid labeling and detection systems were purchased from Amersham Pharmacia Biotech.
Methods
General methods of molecular genetics were practiced according to Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989).
Chromosomal DNA from Corynebacterium aquaticum AKU611 (FERM BP-6448) was isolated using a Genome Isolation Kit (BIO101).
Polymerase chain reaction (PCR) was performed with a thermal cycler (PE Biosystems). Degenerate PCR primers were synthesized by the phosphoramidite method using an Applied Biosystems Model 381A automatic synthesizer (PE Biosystems).
Nucleotide sequence analysis was performed using the dideoxy chain-termination method (Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)). A Taq dye primer sequencing kit was used with an autosequencer (DNA Sequencer 377A, PE Biosystems).
Levodione reductase activity was determined by spectrophotometrically measuring the levodione-dependent decrease in the absorbance of NADH content at 340 nm. A standard 2.5 ml assay mixture contained 5 xcexcmole of levodione (final concentration, 2.0 mM), 0.8 xcexcmole of NADH, 500 xcexcmole of potassium phosphate buffer (pH 7.0), and the enzyme. One unit of the enzyme activity was defined as the amount of enzyme that catalyzes oxidation of 1 xcexcmole of NADH per minute.
Quantitative analysis of the levodione and actinol content was performed with a Shimadzu model GC-14B GC equipped with a flame ionization detector using a type HR-20M capillary column (0.25 mm by 30 m; Shinwa Chemical Industries) at 160xc2x0 C. (isothermal) and He as the carrier gas at a flow rate of 1 ml/min. Under these conditions, levodione, actinol, and (4S,6R)-hydroxy-2,2,6-trimethylcyclohexanone (a diastereomer of actinol) eluted at 6.8, 15.6, and 15.9 minutes, respectively.
Purification of levodione reductase was performed according to the procedures disclosed in European Patent Application No. 99102037.1 filed on Feb. 1, 1999, and included the use of any of the following, alone or in combination: fractionation with precipitants (e.g., ammonium sulfate, polyethylene glycol and the like), ion exchange chromatography, absorption chromatography, gel-filtration chromatography, gel electrophoresis, and salting out and dialysis. The purified enzyme was digested with lysyl endopeptidase (Wako Pure Chemicals) under the conditions described in Appl. Environ. Microbiol. 62, 2303-2310, 1996. The peptides were separated by reverse-phase high-performance liquid chromatography on a xcexcRPC C2/C18 column (Amersham Pharmacia Biotech) connected to a Smart system (microscale protein purification system; Amersham Pharmacia Biotech). The peptides were eluted with a linear 0 to 80% acetonitrile gradient containing 0.1% trifluoroacetic acid.
A partial amino acid sequence was determined by automated Edman degradation with a model 476A pulsed liquid protein sequencer (PE Biosystems) as described previously (Biosci. Biotechnol. Biochem. 62, 280-285, 1998). The partial amino acid sequence obtained was compared with the sequences of proteins stored in the SWISS-PROT (release 37.0+/06-June 14, 1999), PIR (release 60.0, March 1999), and PRF (release 99-May 5, 99) protein databases. Sequence alignment was performed by using the Blast (J. Mol. Biol. 215, 403-410, 1990) and Fasta (Proc. Natl. Acad. Sci. USA 85, 2444-2448, 1988) programs.